Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia.
ABSTRACT: Background: This study's aim was to investigate a large cohort of dystonia patients for pathogenic and rare variants in the ATM gene, making use of a new, cost-efficient enrichment technology for NGS-based screening. Methods: Single molecule Molecular Inversion Probes (smMIPs) were used for targeted enrichment and sequencing of all protein coding exons and exon-intron boundaries of the ATM gene in 373 dystonia patients and six positive controls with known ATM variants. Additionally, a rare-variant association study was performed. Results: One patient (0.3%) was compound heterozygous and 21 others were carriers of variants of unknown significance (VUS) in the ATM gene. Although mutations in sporadic dystonia patients are not common, exclusion of pathogenic variants is crucial to recognize a potential tumor predisposition syndrome. SmMIPs produced similar results as routinely used NGS-based approaches. Conclusion: Our results underline the importance of implementing ATM in the routine genetic testing of dystonia patients and confirm the reliability of smMIPs and their usability for germline screenings in rare neurodegenerative conditions.
Project description:Background: Dystonia-24 (DYT24) is a monogenic autosomal dominant dystonia caused by mutations in the gene ANO3, which has shown phenotypic and genotypic heterogeneity according to previous reports. Objective: To screen and identify ANO3 mutations in a cohort of patients with dystonia in China and to expand the spectrum of DYT24. Methods: This study screened ANO3 mutations in 187 Chinese dystonia patients using next-generation sequencing (NGS). In silico investigations were conducted in detected ANO3 variants and co-segregation analysis was carried out if applicable. The effects of identified variants were classified according to the standards and guidelines of the American College of Medical Genetics and Genomics (ACMG). Results: Four different variants were identified in four unrelated dystonia patients, including three missense variants [c.1789G>C (p.V600L), c.182A>C (p.E61A), c.787A>G (p.M263V)] and one splice site change (c.1714-3T>C). The novel missense mutation c.1798G>C (p.V600L), identified in a teenaged girl with generalized dystonia, showed high pathogenicity and was classified as "likely pathogenic" according to ACMG guidelines. Of note, she responded well to deep brain stimulation. Conclusion: Our study helps expand the mutational and clinical spectrum of DYT24 due to ANO3 mutations by further reporting four variants. Rare ANO3 variants appear to represent an uncommon cause of dystonia in China.
Project description:To identify the cause of cervical dopa-responsive dystonia (DRD) in a Muslim Indian family inherited in an apparently autosomal recessive fashion, as previously described in this journal.Previous testing for mutations in the genes known to cause DRD (GCH1, TH, and SPR) had been negative. Whole exome sequencing was performed on all 3 affected individuals for whom DNA was available to identify potentially pathogenic shared variants. Genotyping data obtained for all 3 affected individuals using the OmniExpress single nucleotide polymorphism chip (Illumina, San Diego, CA) were used to perform linkage analysis, autozygosity mapping, and copy number variation analysis. Sanger sequencing was used to confirm all variants.After filtering of the variants, exome sequencing revealed 2 genes harboring potentially pathogenic compound heterozygous variants (ATM and LRRC16A). Of these, the variants in ATM segregated perfectly with the cervical DRD. Both mutations detected in ATM have been shown to be pathogenic, and ?-fetoprotein, a marker of ataxia telangiectasia, was increased in all affected individuals.Biallelic mutations in ATM can cause DRD, and mutations in this gene should be considered in the differential diagnosis of unexplained DRD, particularly if the dystonia is cervical and if there is a recessive family history. ATM has previously been reported to cause isolated cervical dystonia, but never, to our knowledge, DRD. Individuals with dystonia related to ataxia telangiectasia may benefit from a trial of levodopa.
Project description:BACKGROUND:We aimed to determine the mutation yield and clinical applicability of "molecular autopsy" following sudden arrhythmic death syndrome (SADS) by validating and utilizing low-cost high-throughput technologies: Fluidigm Access Array PCR-enrichment with Illumina HiSeq 2000 next generation sequencing (NGS). METHODS:We validated and optimized the NGS platform with a subset of 46 patients by comparison with Sanger sequencing of coding exons of major arrhythmia risk-genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, RYR2). A combined large multi-ethnic international SADS cohort was sequenced utilizing the NGS platform to determine overall molecular yield; rare variants identified by NGS were subsequently reconfirmed by Sanger sequencing. RESULTS:The NGS platform demonstrated 100% sensitivity for pathogenic variants as well as 87.20% sensitivity and 99.99% specificity for all substitutions (optimization subset, n?=?46). The positive predictive value (PPV) for NGS for rare substitutions was 16.0% (27 confirmed rare variants of 169 positive NGS calls in 151 additional cases). The overall molecular yield in 197 multi-ethnic SADS cases (mean age 22.6?±?14.4?years, 68% male) was 5.1% (95% confidence interval 2.0-8.1%), representing 10 cases carrying pathogenic or likely pathogenic risk-mutations. CONCLUSIONS:Molecular autopsy with Fluidigm Access Array and Illumina HiSeq NGS utilizing a selected panel of LQTS/BrS and CPVT risk-genes offers moderate diagnostic yield, albeit requiring confirmatory Sanger-sequencing of mutational variants.
Project description:Objective:We investigated the contribution to sporadic focal epilepsies (FE) of ultrarare variants in genes coding for the components of complexes regulating mechanistic Target Of Rapamycin (mTOR)complex 1 (mTORC1). Methods:We collected genetic data of 121 Italian isolated FE cases and 512 controls by Whole Exome Sequencing (WES) and single-molecule Molecular Inversion Probes (smMIPs) targeting 10 genes of the GATOR1, GATOR2, and TSC complexes. We collapsed "qualifying" variants (ultrarare and predicted to be deleterious or loss of function) across the examined genes and sought to identify their enrichment in cases compared to controls. Results:We found eight qualifying variants in cases and nine in controls, demonstrating enrichment in FE patients (P = 0.006; exact unconditional test, one-tailed). Pathogenic variants were identified in DEPDC5 and TSC2, both major genes for Mendelian FE syndromes. Interpretation:Our findings support the contribution of ultrarare variants in genes in the mTOR pathway complexes GATOR and TSC to the risk of sporadic FE and a shared genetic basis between rare and common epilepsies. The identification of a monogenic etiology in isolated cases, most typically encountered in clinical practice, may offer to a broader community of patients the perspective of precision therapies directed by the underlying genetic cause.
Project description:Next-generation sequencing (NGS) hereditary pan-cancer panel testing can identify somatic variants, which exhibit lower allele frequencies than do germline variants and may confound hereditary cancer predisposition testing. This analysis examined the prevalence and characteristics of likely-somatic variants among 348,543 individuals tested using a clinical NGS hereditary pan-cancer panel. Variants showing allele frequencies between 10% and 30% were interpreted as likely somatic and identified in 753 (0.22%) individuals. They were most frequent in TP53, CHEK2 and ATM, commonly as C-to-T transitions. Among individuals who carried a likely-somatic variant and reported no personal cancer history, 54.2% (78/144) carried a variant in TP53, CHEK2 or ATM. With a reported cancer history, this percentage increased to 81.1% (494/609), predominantly in CHEK2 and TP53. Their presence was associated with age (OR=3.1, 95% CI 2.5, 3.7; p<0.001) and personal history of cancer (OR=3.3, 95% CI 2.7, 4.0; p<0.001), particularly ovarian cancer. Germline ATM pathogenic variant carriers showed significant enrichment of likely-somatic variants (OR=2.8, 95% CI 1.6, 4.9; p?=?0.005), regardless of cancer status. The appearance of likely-somatic variants is consistent with clonal hematopoiesis, possibly influenced by cancer treatment. These findings highlight the precision required of diagnostic laboratories to deliver accurate germline testing results.
Project description:Pathogenic variants in known breast cancer (BC) predisposing genes explain only about 30% of Hereditary Breast Cancer (HBC) cases, whereas the underlying genetic factors for most families remain unknown. Here, we used whole-exome sequencing (WES) to identify genetic variants associated to HBC in 17 patients of Brazil with familial BC and negative for causal variants in major BC risk genes (BRCA1/2, TP53, and CHEK2 c.1100delC). First, we searched for rare variants in 27 known HBC genes and identified two patients harboring truncating pathogenic variants in ATM and BARD1. For the remaining 15 negative patients, we found a substantial vast number of rare genetic variants. Thus, for selecting the most promising variants we used functional-based variant prioritization, followed by NGS validation, analysis in a control group, cosegregation analysis in one family and comparison with previous WES studies, shrinking our list to 23 novel BC candidate genes, which were evaluated in an independent cohort of 42 high-risk BC patients. Rare and possibly damaging variants were identified in 12 candidate genes in this cohort, including variants in DNA repair genes (ERCC1 and SXL4) and other cancer-related genes (NOTCH2, ERBB2, MST1R, and RAF1). Overall, this is the first WES study applied for identifying novel genes associated to HBC in Brazilian patients, in which we provide a set of putative BC predisposing genes. We also underpin the value of using WES for assessing the complex landscape of HBC susceptibility, especially in less characterized populations.
Project description:Recent development of next-generation DNA sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We applied NGS to study an A-T patient in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. The 160-kb ATM genomic region was divided into 31 partially overlapping fragments of 4-6?kb and amplified by long-range PCR in the patient and mother, who carried the same mutation by segregation. We identified six intronic variants that were shared by the two genomes and not reported in the dbSNP(132) database. Among these, c.1236-405C>T located in IVS11 was predicted to be pathogenic because it affected splicing. This mutation creates a cryptic novel donor (5') splice site (score 1.00) 405?bp upstream of the exon 12 acceptor (3') splice site. cDNA analysis showed the inclusion of a 212-bp non-coding 'pseudoexon' with a premature stop codon. We validated the functional effect of the splicing mutation using a minigene assay. Using antisense morpholino oligonucleotides, designed to mask the cryptic donor splice-site created by the c.1236-405C>T mutation, we abrogated the aberrant splicing product to a wild-type ATM transcript, and in vitro reverted the functional ATM kinase impairment of the patients' lymphoblasts. Resequencing is an effective strategy for identifying rare splicing mutations in patients for whom other mutation analyses have failed (DHPLC, MLPA, or cDNA sequencing). This is especially important because many of these patients will carry rare splicing variants that are amenable to antisense-based correction.
Project description:Ovarian cancer represents the most common gynaecological malignancy and has the highest mortality of all female reproductive cancers. It has a rare predilection to develop brain metastases (BM). In this study, we evaluated the mutational profile of ovarian cancer metastases through Next-Generation Sequencing (NGS) with the aim of identifying potential clinically actionable genetic alterations with options for small molecule targeted therapy. Library preparation was conducted using Illumina TruSight Rapid Capture Kit in combination with a cancer specific enrichment kit covering 94 genes. BRCA-mutations were confirmed by using TruSeq Custom Amplicon Low Input Kit in combination with a custom-designed BRCA gene panel. In our cohort all eight sequenced BM samples exhibited a multitude of variant alterations, each with unique molecular profiles. The 37 identified variants were distributed over 22 cancer-related genes (23.4%). The number of mutated genes per sample ranged from 3 to 7 with a median of 4.5. The most commonly altered genes were BRCA1/2, TP53, and ATM. In total, 7 out of 8 samples revealed either a BRCA1 or a BRCA2 pathogenic mutation. Furthermore, all eight BM samples showed mutations in at least one DNA repair gene. Our NGS study of BM of ovarian carcinoma revealed a significant number of BRCA-mutations beside TP53, ATM and CHEK2 mutations. These findings strongly suggest the implication of BRCA and DNA repair malfunction in ovarian cancer metastasizing to the brain. Based on these findings, pharmacological PARP inhibition could be one potential targeted therapeutic for brain metastatic ovarian cancer patients.
Project description:Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904_906delGAG/c.907_909delGAG; p.Glu302del/Glu303del; ?E 302/303) near the carboxyl-terminus of torsinA, a member of the AAA(+) protein family that localizes to the endoplasmic reticulum lumen and nuclear envelope. This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinA?E, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients: R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinA?E and wild-type (wt) torsinA to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinA?E protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia.
Project description:PURPOSE:To date, there has not been a large, systematic evaluation of the prevalence of germline risk variants in urothelial carcinoma (UC). METHODS:We evaluated the frequency of germline pathogenic and likely pathogenic variants in 1038 patients with high-risk UC who underwent targeted clinical germline testing. Case-control enrichment analysis was performed to screen for pathogenic variant enrichment in 17 DNA repair genes in 1038 UC patients relative to cancer-free individuals. RESULTS:Among 1038 patients with UC, the cumulative frequency of patients with pathogenic variants was 24%; 18.6% of patients harbored ?1 actionable germline variant with preventive or therapeutic utility. MSH2 (34/969, 3.5%) and BRCA1/2 (38/867, 4.4%) germline variants had the highest frequency. Germline variants in DNA damage repair genes accounted for 78% of pathogenic germline variants. Compared to the cancer-free cohort, UC patients had significant variant enrichment in MSH2 (odds ratio [OR]: 15.4, 95% confidence interval [CI]: 7.1-32.7, p?<?0.0001), MLH1 (OR: 15.9, 95% CI: 4.4-67.7, p?<?0.0001), BRCA2 (OR: 5.7, 95% CI: 3.2-9.6, p?<?0.0001), and ATM (OR: 3.8, 95% CI: 1.8-8.3, p?=?0.02). CONCLUSION:In this study, 24% of UC patients harbored pathogenic germline variants and 18.6% had clinically actionable variants. MLH1 and MSH2 were validated as UC risk genes while ATM and BRCA2 were highlighted as potential UC predisposition genes. This work emphasizes the utility of germline testing in selected high-risk UC cohorts.